To determine whether the FE65 proteins, FE65 and FE65L1, are required to sort nascent APP to the secretory/fast axonal transport pathway, we measured nascent, presynaptic APP in the retinal ganglion cell (RGC) terminals in the first-class colliculus (SC) of WT and FE65/FE65L1 DKO mice. reported (Hoey 2009, Marcello 2007, Lesne 2005, Hoe 2009). These disagreements may result from activation protocols that target specific swimming pools of NMDARs, e.g. synaptic versus extrasynaptic NMDARs. Some studies statement that NMDAR activation raises APPs secretion and reduces A secretion (Hoey 2009, Marcello 2007), effects that have been attributed to synaptic NMDAR activation (Hoey 2009). In contrast, activation of extrasynaptic NMDARs was recently reported to increase A production (Bordji 2010). The FE65 family of APP-binding proteins (FE65, FE65L1 and FE65L2) modulate APP processing (King & Scott Turner 2004). FE65 or FE65L1 protein overexpression in cell lines increase A production and these changes were attributed to improved trafficking of APP into the endocytic pathway (Guenette 1999, Chang 2003, Sabo 1999). Consistent with these findings, cerebral A levels were reduced in FE65/FE65L1 DKO mice (Guenette 2006). Variations in neuronal APP processing between crazy type (WT) and FE65/FE65L1 double knockout (DKO) mice NVP-BHG712 may be responsible for the reduction in cerebral A levels observed in FE65/FE65L1 DKO mice since neuron ethnicities founded from Tg2576 mice lacking the 97KDa FE65 isoform secrete less A40 and A42 than control Tg2576 neurons (Wang 2004). To further assess whether and how FE65 proteins influence APP trafficking and processing in neurons, we examined the effects of FE65 protein knockout on APP transport 1995). NMDA, AMPA, MK-801, CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), calpeptin, lactacystin and MG132 were purchased from Calbiochem. In our ethnicities, approximately 10% neuronal cell death was observed for neurons treated with 10 M NMDA (24 hr) as assessed by lactate dehydrogenase launch (data not demonstrated). Fast Axonal transport of APP Metabolic labeling of neuronal proteins was performed by unilateral intraocular injection of 35S-Methionine/Cysteine (100 Ci/mouse) in nine (n=4 WT and n=5 FE65/FE65L1 DKO; 4.5C6.5 months of age) mice under avertin (0.5 mg/g i.p.) anesthesia. Animals were euthanized 4.5 hrs post-injection and brains were dissected. APP was immunoprecipitated from your superior colliculus using the A8717 APP C-terminal antibody (Sigma). Immunoprecipitates were separated on 4C12% Nupage gels with Bis-Tris operating buffer; transferred to PVDF filters; and quantitated using storage phosphor imaging. All animal experiments were performed in accordance with the MGH Institutional Animal Care and Use Committee. Semi-quantitative reverse transcription-PCR Total RNA from main cultured mouse neurons and mind cells was isolated using RNAzol (Gibco). RNA integrity was confirmed by detection of the 28S and 18S ribosomal RNA bands. RNA was also confirmed to have no detectable genomic DNA contamination by PCR in the absence of reverse transcriptase. RNA (0.5 g) was reverse transcribed into cDNA (TaqMan Reverse Transcription kit, Applied Biosystems) and each APP isoform was amplified using experimentally optimized cycles that allowed assessment between genotypes within the linear amplification range Rabbit Polyclonal to ARC (PCR expert mix, Qiagen). Oligonucleotide primers for RT-PCR of APP770, APP751, APP695, -actin and APP exon 15 were previously reported (Lesne et al. 2005, Konig 1992). PCR products were separated on 2% agarose gels and visualized with NVP-BHG712 ethidium bromide staining. Relative intensities of PCR bands were analyzed using the Gel Doc 1000 video-imaging system (Bio-Rad). Western blot analysis and antibodies Main neurons and embryonic brains were lysed in RIPA buffer comprising 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS) and protease inhibitor cocktail containing 1 mM EDTA (Roche) on snow for 15 mins. Protein samples for SDS-PAGE were prepared in NuPAGE LDS sample buffer comprising -mercaptoethanol (2.5%) and separated on 4C12% NuPAGE Novex Bis-Tris or 3C8% NuPAGE Novex Tris-Acetate gels (Invitrogen). NVP-BHG712 For AICD detection, neuronal lysates were harvested directly with SDS-sample buffer comprising protease inhibitors (Kimberly 2005) and separated on 10C20% or 16% tricine gels (Invitrogen). Proteins were transferred to PVDF (Millipore) membranes for Western blot analyses. Both A8717 (Sigma) and C66 (Huttunen 2007) were raised against the last 20 amino acid of the APP C-terminus. The epitope for CT695 (Zymed) is the last 22 amino acids of APP. 22C11 (Chemicon) recognizes amino acids 66C81 of the N-terminus of APP and APLP2. R7 (Refolo 1989) and abdominal12269 (Abcam) are specific for KPI-APP. APP phosphorylated in the Thr668 residue (pThr668APP) was recognized having a phosphoAPP (Thr668) antibody (Cell Signaling). APLP1 and APLP2 NVP-BHG712 were recognized with antibodies raised to the C-termini of APLP1 and APLP2 that do not cross-react with APP (Myre 2009). Immunoreactive bands on Western blots were recognized with enhanced chemiluminescence reagents.
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